Genomic profiling of the two types of sporadic CRCs revealed significant differences in the extent and distribution of CN alterations in the cancer genome.. Combining the data of gen-ome
Trang 1R E S E A R C H Open Access
Molecular profile and copy number analysis of sporadic colorectal cancer in Taiwan
Chien-Hsing Lin1†, Jen-Kou Lin2†, Shih-Ching Chang2†, Ya-Hui Chang1, Hwey-May Chang2, Jin-Hwang Liu3,
Ling-Hui Li4, Yuan-Tsong Chen4, Shih-Feng Tsai1,5 and Wei-Shone Chen2*
Abstract
Background: Colorectal cancer (CRC) is a major health concern worldwide, and recently becomes the most
common cancer in Asia The case collection of this study is one of the largest sets of CRC in Asia, and serves as representative data for investigating genomic differences between ethnic populations We took comprehensive and high-resolution approaches to compare the clinicopathologic and genomic profiles of microsatellite instability (MSI)
vs microsatellite stability (MSS) in Taiwanese sporadic CRCs
Methods: 1,173 CRC tumors were collected from the Taiwan population, and sequencing-based microsatellite typing assay was used to determine MSI and MSS Genome-wide SNP array was used to detect CN alterations in 16 MSI-H and 13 MSS CRCs and CN variations in 424 general controls Gene expression array was used to evaluate the effects
of CN alterations, and quantitative PCR methods were used to replicate the findings in independent clinical samples Results: These 1,173 CRC tumors can be classified into 75 high-frequency MSI (MSI-H) (6.4%), 96 low-frequency MSI (8.2%) and 1,002 MSS (85.4%) Of the 75 MSI-H tumors, 22 had a BRAF mutation and 51 showed MLH1 promoter hypermethylation There were distinctive differences in the extent of CN alterations between CRC MSS and MSI-H subtypes (300 Mb vs 42 Mb per genome, p-value < 0.001) Also, chr7, 8q, 13 and 20 gains, and 8p and 18 losses were frequently found in MSS but not in MSI-H Nearly a quarter of CN alterations were smaller than 100 kb, which might have been missed in previous studies due to low-resolution technology 514 expressed genes showed CN differences between subtypes, and 271 of them (52%) were differentially expressed
Conclusions: Sporadic CRCs with MSI-H displayed distinguishable clinicopathologic features, which differ from those
of MSS Genomic profiling of the two types of sporadic CRCs revealed significant differences in the extent and
distribution of CN alterations in the cancer genome More than half of expressed genes showing CN differences can directly contribute to their expressional diversities, and the biological functions of the genes associated with CN changes in sporadic CRCs warrant further investigation to establish their possible clinical implications
Background
Colorectal cancer (CRC) is one of the major leading
causes of cancer deaths around the world, and is the
most common cancer in Taiwan [1] Two different
genetic pathways have been described for tumorigenesis
of CRC The most frequent pathway is the chromosomal
instability pathway characterized by alterations in tumor
suppressor genes and oncogenes, including APC, TP53
and K-ras [2,3] On the other hand, 10-15% of all cases
of CRC show microsatellite instability (MSI), which are resulted from a germline mutation in the mismatch repair (MMR) system or somatic hypermethylation of the promoter region of the MLH1 gene [4] Tumors with MMR deficiency exhibited frequent errors in microsatellite DNA, short segments of DNA containing tandem repeats of mono-, di- or trinucleotides [5] The high-frequency MSI (MSI-H) CRCs have unique clinico-pathologic features, such as right-sided, mucinous or poorly differentiated, and stable chromosomal status in the tumors [6]
About 80% of MSI tumors have a near-diploid karyo-type and a distinct genetic alteration distinguishable from those of microsatellite stable (MSS) cancers [7-10]
* Correspondence: wschen@vghtpe.gov.tw
† Contributed equally
2
Division of Colon and Rectal Surgery, Department of Surgery, Taipei
Veterans General Hospital, Taipei, Taiwan
Full list of author information is available at the end of the article
© 2011 Lin et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
Trang 2Despite the advancement of our understanding of cancer
genetics of CRC, genomic alterations of various subtypes
of CRC have not been fully characterized The copy
num-ber variations (CNVs) can contribute to variable levels of
gene expressions [11], and thus fine-scale copy number
(CN) profiling of cancer can enhance our knowledge
about tumorigenesis Among all somatic mutations,
non-germline CNVs found in the cancer genomes, also
known as copy number alterations/aberrations (CNAs),
are frequently observed, e.g., gains of oncogenes and
losses of tumor suppresser genes [12] Furthermore, the
DNA CN states of CRC cases are related to the response
of drug treatments, e.g., the CNA degree of CRC is
asso-ciated with response to systemic combination
che-motherapy with capecitabine and irinotecan [13]
Previous cytogenetic studies have shown MSS tumors
are characterized with more chromosomal and copy
number aberrations than MSI tumors [14,15], and most
of MSI tumors have a near-diploid karyotype and appear
to follow a genetic pathway distinct from MSS tumors
[9] These studies showed that gain of chromosome 7,
8q, 13 and 20q and loss of chromosome 4q, 8p, 17p and
18q were frequent in CRC MSS tumors [16] Both
pro-files of genome-wide CNA and gene expression have
been used to classify MSS and MSI subtypes of CRC
samples [17] However, previous genome-wide CNA
stu-dies of CRC were limited by the resolution of
compara-tive genomic hybridization (CGH) array technology
(probe distance > 30 kb), thereby subtle CN changes
har-boring cancer-causing variants might be missed
[13,17,18] As genomic technology advances, high-density
single-nucleotide polymorphism (SNP) array can be used
to genotype a huge number of SNPs and detect CN
changes on the genomic scale In the current study, we
have applied Affymetrix SNP 6.0 array (Affymetrix, CA,
USA), with its median inter-probe distance of less than
700 bp, to detect CNAs in CRC cancer genome of clinical
samples As compared to other reports on the CRC
can-cer genome using the CGH arrays, we have achieved a
much improved resolution Molecular karyotype profiling
of the two subtypes of sporadic CRCs revealed significant
differences in the extent and distribution of CN
altera-tions in the cancer genome Combining the data of
gen-ome-wide CNAs and Illumina Human Ref-8 gene
expression array (Illumina, CA, USA), CNAs might
sig-nificantly contribute to the expressional levels of genes,
more than half of which were differently expressed
between CRC MSI-H and MSS
Materials and methods
Clinical patients and tumor tissues
A total of 1,543 colorectal cancer patients who
under-went surgeries in Taipei Veterans General Hospital from
January 2000 to December 2007 were included The study was approved by the Institutional Review Board of the Taipei Veterans General Hospital, and written informed consent for tissue collection was obtained from all patients Patient with preoperative chemoradiother-apy, or emergent operative procedure, or death within
30 postoperative days, or evidence of familial adenoma-tous polyposis were excluded from this study Clinical information was recorded prospectively and stored in a database This included: (i) age, sex, personal and family history, and (ii) tumor size, location, gross appearance, TNM stage, differentiation and pathological prognostic features Tumors were meticulously dissected, with sam-ples collected from the 4 tumor quadrants to explore intratumoral heterogeneity The corresponding normal mucosa, at least 10 cm away from the primary tumor edge, was collected Tissue fragments were immediately frozen in liquid nitrogen and stored at -70°C Sections of cancerous and collateral tissues were reviewed and ana-lyzed by a senior gastrointestinal pathologist blinded to patient outcomes Disease stage was determined with the TNM classification of the International Union Against Cancer [19] The pathological factors analyzed included lymphovascular invasion, invasive tumor pattern, grade
of differentiation, mucin production and intratumoral lymphocyte infiltration These pathological features were defined by the College of American Pathologists consen-sus statement [20]
Microsatellite Instability Analysis
High-molecular-weight genomic DNA from each tumor and from corresponding normal tissue was purified using the QIAamp Tissue kit (QIAGEN GmbH, Germany) Yield and purity were determined by electrophoresis on 0.8% agarose gel and spectrophotometric absorbance at
260 nm According to international criteria for determi-nation of MSI,5 five reference microsatellite markers were used: D5S345, D2S123, BAT25, BAT26, and D17S250 Primer sequences were obtained from Gen-Bank (http://www.gdb.org) Detection of MSI was per-formed as previously described [20,21] Briefly, DNA was amplified using fluorescent polymerase chain reaction (PCR) PCR products were denatured and analyzed by electrophoresis on 5% denaturing polyacrylamide gels, and results were analyzed using GeneScan Analysis soft-ware (Applied Biosystems, CA, USA) Tumor samples that exhibited allele peaks different from the correspond-ing normal sample(s) were classified as MSI for that par-ticular marker Samples with≥ 2 MSI of 5 markers were defined as MSI-H, those with only one MSI of 5 markers were defined as low-frequency MSI (MSI-L) and others without evidence of MSI were classified as MSS Analyses were performed twice if results were ambiguous
Trang 3Immunohistochemistry (IHC) staining for MLH1,
MSH2, MSH6 and PMS2 were done for cases with
MSI-H Paraffin-embedded tissue sections (4μm thickness)
were stained with antibodies for MLH1 (1:10 dilution,
Pharmingen), MSH2 (1:200, Oncogene Research
Pro-ducts), MSH6 (1:300, Transduction Laboratories) and
PMS2 (C20) (1:400, Santa Cruz Biotechnology) Negative
control slides were made without the primary antibody
BRAF mutation and MLH1 methylation analysis
To detect BRAF mutation, DNA from tumor tissue was
amplified and sequenced with primers described in
pre-vious studies [22] Briefly, the extracted DNA was
selec-tively amplified by PCR in a DNA thermocycler A
negative control containing no DNA template was
included for each PCR amplification round The PCR
products were analyzed by an automated sequencer
(ABI Prism 3100 Genetic Analyzer; Applied Biosystems)
Each sample was sequenced on both sense and antisense
strands Each mutation was confirmed by a second
sequencing procedure on new PCR products
Methyla-tion of the MLH1 promoter was determined using a
methylation-specific PCR method DNA was treated
with sodium bisulfite, which converts unmethylated
cytosine to uracil, yet leaves methylated cytosine
unchanged, and subjected to amplification with
methy-lated- and unmethymethy-lated-specific primers, respectively
[23]
Flow Cytometry for DNA Ploidy
703 of 1,173 tumors were available to examine the status
of DNA ploidy using flow cytometry by following the
method of Dressler et al [24] The DNA index (DI),
representing the ratio of the mean fluorescence intensity
of the G0G1peak of the tumor cell population to that of
the normal diploid population, was used to quantitate
DNA ploidy Specimens were considered diploid (DI = 1)
if they had a single G0G1 peak and aneuploid (DI≠ 1) if
they exhibited two or more discrete peaks, including
abnormal G0G1peaks (each peak equivalent to the
fluor-escence of at least 20% of the total sample nuclei) and a
corresponding G2M peak Samples with coefficients of
variation > 8% were excluded from further analysis [21]
Tumors with both diploid and aneuploid subpopulations
were classified as having DNA aneuploidy The mean
coefficients of variation were 6.4% and 2.4% in tumor
tis-sues and normal colon mucosa, respectively
High-density SNP array and data analysis
A total of 500 ng of genomic DNA of 16 MSI-H and 13
MSS CRC samples was subjected to SNP genotyping
using genome-wide Affymetrix Human SNP 6.0 array
according to the manufacturer’s instructions Genotyping
was performed by the National Genotyping Center at Academia Sinica, Taipei, Taiwan (http://ngc.sinica.edu tw) This array contains 1.8 millions markers widely dis-tributing in human genome After standard Affymetrix quantile normalization, the intensity data was analyzed using Genotyping Console (GTC) software v.3.0.1 (Affy-metrix) with default parameters of hidden-Markov model (HMM) to identify CN-changed regions [25] PennCNV [26] and Partek Genome Suite (Partek Inc., MO, USA) software were additionally used to reconfirm CN altera-tions identified by GTC software CNA predicted by PennCNV and Partek software with default HMM para-meters are 91.6% and 89.8% concordant with those of GTC software In consideration of CN-changed regions with at least 20 consecutive probes, we found that all these CNA identified are 100% overlapped with those defined by either PennCNV or Partek software, implying these CNAs were highly reliable for the following analysis
Quantitative genomic PCR
CN changes of selected genes, including epidermal growth factor receptor (EGFR), deleted colon cancer (DCC) and calcium-dependent membrane-binding protein 1 (CPNE1), were verified by using quantitative genomic PCR experiments Primer Express Software version 3.0 (Applied Biosystems) was applied to design PCR primers for the selected target genes Quantitative genomic PCR were per-formed using the ABI StepOne Plus system (Applied Bio-systems) PCR reactions were prepared using the Power SYBR-Green PCR reagent kit (Applied Biosystems), and 2.5 ng genomic DNA was used in each reaction qPCR conditions were as follows: initial denaturation at 94°C for
3 minutes, followed by 40 cycles of denaturation at 94°C for 15 seconds, and combined annealing and extension at 60°C for 60 seconds The fluorescence signal was detected
in real time during the qPCR procedure The primer pair for the long interspersed nuclear elements 1 sequence was used for normalization The mean estimated CN was cal-culated from triplicate PCR reactions for each individual
Whole-genome gene expression analysis
RNA samples of 16 MSI-H and 13 MSS tumors (identical cases as used in SNP array analysis) were prepared using Qiagen’s RNAeasy kit (Qiagen), and then were assayed using the Agilent Systems Bioanalyzer (Agilent Technol-ogies, CA, USA) to ensure that high-quality RNA was used for the gene expression array experiments The Illu-mina TotalPrep RNA amplification kit (Ambion, TX, USA) was used to amplify and generate biotinylated RNA Illumina Human Ref-8 V3 arrays were processed and scanned at medium PMT settings as recommended
by the manufacturer, and were analyzed using GenomeS-tudio software (Illumina) After subtracting background,
Trang 4array data was normalized using the quantile method,
and detection p-value < 0.01 was used to ensure that only
expressed genes were used in the following analyses
Statistical analysis
All results in the text and tables are given as means ±
stan-dard deviation In clinical analyses, categorical variables
were analyzed using a chi-square test with Yates’
correc-tion, and comparisons of quantitative variables between
groups were performed based on Student’s t-test In
geno-mic data analysis, CNA frequency comparisons between
CRC MSS and MSI-H subtypes were carried out by using
Fisher’s exact test, and t-test was applied in comparing
expressional levels of each transcript between CRC
sub-types SAS/STAT (SAS Institute, NC, USA) program was
used to carry out all statistical analyses
Results
A total of 1,543 CRCs were recruited in Taiwan
popula-tion from 2000 to 2007 as shown in Figure 1 To focus on
sporadic CRC cases for the clinicopathologic and genomic
analyses, 370 (24.0%) meeting the Revised Bethesda
cri-teria [27], defined patients having CRC familiar history,
were excluded, and the remaining 1,173 patients were
sporadic CRC cases There were 785 (66.9%) males and
388 (33.1%) females in these sporadic CRC patients
Tumors were found in right-side colon in 294 patients
(25.1%), left-side colon in 478 patients (40.8%), and in the
rectum in 401 patients (34.2%) There were 159 patients
(13.6%) with stage I cancers, 395 patients (33.7%) with
stage II cancers, 407 patients (34.7%) with stage III cancers
and 212 patients (18.1%) with stage IV cancers Based on
microsatellite instability analysis, among the 1,173 tumors
analyzed, 75 (6.4%) were MSI-H, 96 (8.2%) were MSI-L,
and 1,002 (85.4%) were MSS Interestingly, 48 out of the
75 MSI-H tumors (64%) were located in the right colon;
67% had stage I or II disease; 60% were female and 24%
were poorly or mucinous differentiated (Table 1) In
con-trast to the clinopathologic features of MSI-H tumors,
MSS/MSI-L showed left sided predominant, less mucinous
or poorly differentiation and more advanced disease
Methylation of the MLH1 gene promoter and BRAF
gene mutations were analyzed for all MSI-H tumors Of
the 75 MSI-H tumors, 22 (29.3%) had a BRAF mutation
and 51 (68%) showed hypermethylation of the MLH1 gene
promoter Immunohistochemical (IHC) stains for MLH1,
MSH2, MSH6 and PMS2 proteins were carried out for 70
cases with MSI-H tumors whose samples were available
(Figure 2) As shown in Figure 1, 47 of 70 (67.1%) MSI-H
tumors showed abnormalities with IHC analysis for at
least one MMR protein The majority (n = 40, 57.1%) lost
MLH1 protein expression, followed by MSH2 protein (n =
8, 11.4%) Among the 40 tumors with no detectable
MLH1 protein expression, 32 had hypermethylation of the
promoter (80%) and 17 had BRAF mutation (42.5%) Five MSI-H tumors had no expression of either MSH6 or PMS2 protein, and 23 cases (32.9%) had detectable expres-sions of all four MMR proteins (Figure 1)
Of the 703 tumors, including 51 MSI-H and 652 MSI-L/ MSS, available for the status of DNA ploidy, 231 showed DNA diploid (32.9%) We found that 70.2% of MSI-L/MSS tumors showed DNA aneuploidy, but only 27.5% of
MSI-H tumors showed DNA aneuploidy To molecularly char-acterize chromosomal aberrations at a high resolution (≤
20 kb) and compare the genomic features between the MSI-H and MSS subtypes, Affymetrix SNP 6.0 array was applied to detect genome-wide CNAs in 16 MSI-H tumors with both MLH1 hypermethylation and BRAF mutation, and compared to the genomic profiles of 13 MSS CRC tumors To identify reliable CN changes, we only included CN-changed regions covering more than 20 probes, and these CNAs were also called by PennCNV and Partek CNV calling software (algorithm-independent) As a con-trol, the CNV profile of Taiwanese population was based
on 434 general controls from Han Chinese Cell and Gen-ome Bank that were genotyped using Affymetrix SNP 6 array [28] This data provides useful information, at the population scale, the common variation of genomic struc-ture in the Taiwanese study subjects A total of 399 CNV regions were identified in this population (Dr Y.-T Chen, unpublished data), the average size of the CNV regions was 350 kb (covering a total of 4.66% of the human gen-ome), and 372 (93.23%) were reported in the database of genomic variants (http://projects.tcag.ca/variation/) As shown in Figure 3, the whole-genome CNV patterns of the two CRC subtypes were grossly different DNA CN gain in chr7, 8q, 13 and 20 and loss in chr4q, 8p and 18 were frequently found in MSS but not in MSI-H tumors Consisting with previous studies, the chromosomal struc-tures of CRCs with microsatellite instability were similar
to those of normal controls [9] (Figure 3) There were dis-tinctive differences in the number of CNAs between CRC MSS and MSI-H subtypes (Figure 4a, 439 vs 63 per gen-ome, p-value = 0.0005), and the average size of CNAs per genome of MSS tumor was larger than that of MSI-H tumor (Figure 4b, 300 Mb vs 42 Mb, p-value = 0.001) The majority of CNAs (> 80%) found in CRC cases was smaller than 500 kb, and nearly a quarter of CN alterations were smaller than 100 kb, which might have been missed in the previous studies due to low-resolu-tion technologies (Addilow-resolu-tional File 1) Therefore, CNA frequencies of some DNA segments in this study were higher than those from previous studies (14) 13,279 protein-coding genes and 557 microRNA were affected
by CN changes in these CRC samples, of which 1,434 genes (10.8%) and 35 microRNAs (6.3%) were related to CNVs observed in the general Taiwanese population To identify genes harboring the CRC subtype-common and/
Trang 5or specific CN changes, the gene-based CNA frequency
of MSS and MSI-H subtypes were compared as shown
in Figure 5a 1,515 of 13,279 genes (11.4%) were found
to have CN frequency difference between MSS and
MSI-H tumors using Fisher’s exact tests (p-value < 0.05,
Additional File 2), and CNA frequencies of these genes
in MSS tumors were all higher than those in MSI-H
tumors
The CN gain of EGFR gene, a well-known cancer gene
and drug target, was commonly found in CRC MSS
tumors (8 out of 13 samples, 62%) according to
gen-ome-wide CNA analysis To replicate the findings from
the SNP array analysis, we applied qPCR approach to
evaluate the EGFR CN states of independent 48 CRC
MSS and 48 MSI-H samples (Additional File 3) The
CN gain frequency of the independent CRC MSS group was 64.6% (31 of 48) and consisted to that (62%) of the array-based CN analysis, and was higher than overall 14% of CRC MSI-H subtype (n = 64) Furthermore, although CN losses of DCC gene were commonly found
in CRCs in previous studies [29], we observed that this DCC deletions were frequently found in MSS CRCs (46%) but not in MSI-H (0%) Twelve cancer-associated genes were found to show different CN frequencies between CRC subtypes as shown in Table 2 (Fisher’s exact test, p-value < 0.01), but the biological functions
of many identified genes with high CNA frequencies were not fully characterized
Figure 1 Flowchart of genomic study on sporadic CRCs Five reference microsatellite markers are used to classify sporadic CRC cases into microsatellite stability (MSS), low-frequency microsatellite instability (MSI-L), and high-frequency MSI (MSI-H) (shown in Materials and Methods) Immunohistochemistry staining for MLH1, MSH2, MSH6 and PMS2 protein and mutation screening for BRAF gene were done for CRC cases with MSI-H.
Table 1 Clinico-pathological differences between MSI-H and MSI-L/MSS CRCs
Variables MSI-H tumors (N = 75) MSI-L/MSS tumors (N = 1,098) p-value Age 70.2 ± 9.6 70.8 ± 9.2 0.565 Female gender (%) 45(60) 343(31.2) < 0.001 Right colon (%) 48(64.0) 246(22.4) < 0.001 Stage 1,2 (%) 50(66.7) 504(45.9) < 0.001 Mucinous or signet ring adenocarcinoma (%) 18(24.0) 112(11.0) 0.001
Poor differentiated (%) 18(24.0) 57(5.2) < 0.001
Categorical variables were analyzed using a chi-square test with Yate’s correction.
Comparisons of quantitative variables between groups used Student’s t-test.
Trang 6Among 24,526 annotated RefSeq transcripts (18,631
unique genes) of Illumina Human Ref-8 gene expression
array, 12,012 (48.9%) were expressed in tumor tissues
599 and 724 transcripts showed higher- or
lower-expres-sions, respectively, in MSS tumors compared to MSI-H
(Additional File 4) The transcript profiles of nine genes,
as shown in Additional File 5, can be used to well
clas-sify CRC microsatellite status in clinical patients from
Caucasian population [30] Six of them showed
concor-dant expression profiles between Caucasian and Han
Chinese populations, but lower-expressed SFRS6 and
higher-expressed SET genes of CRC MSS tumors in Caucasian were not found in Han Chinese, implying there are subtle population diversities in CRC transcript profiles
Although there were numerous genes affected by CN gains and/or losses in CRC cancer genome, especially in MSS cases, some might not directly contribute to the levels of gene expressions The patterns of differentially-expressed genes between CRC subtypes (two sample t-test with p-value < 0.05) are similar to those of CNA analysis at genome-wide scale (Figure 5b) Only 514 of
Figure 2 Immunohistochemical (IHC) stains for MLH1, MSH2, MSH6 and PMS2 proteins Paraffin-embedded tissue sections (4 μm thickness) of CRC MSI-H and control samples were stained with antibodies for MLH1, MSH2, MSH6 and PMS2 proteins.
Trang 7Figure 3 Whole-genome copy number variation (CNV) pattern of colorectal cancer and general population The CNV frequencies are measured from 16 MSI-H CRCs, 13 MSS CRCs and 434 individuals from general population using Affymetrix SNP 6.0 array Top dots represent the frequencies of CN gains, and bottom dots represent the frequencies of CN losses.
Figure 4 Comparisons of copy number variation patterns between colorectal cancer subtypes (a) the average number of CN-changed regions per genome for MSS, MSI-H and general controls (b) the average size of CN-changed regions per genome for MSS, MSI-H and general controls.
Trang 81,515 showing CNA frequency differences between
sub-types were expressed in tumor tissue, and 271 of them
(52%) were differentially expressed (p-value < 0.05,
Additional File 6), suggesting the CN variations of genes
might underline the expressional diversities between
CRC MSS and MSI-H subtypes For example, CN gains
of CPNE1 genes were found in 8 of 13 MSS but not in MSI-H cases (Additional File 7), and the average CPNE1 expressional levels of MSS tumors was higher then that
of MSI-H (1797.9 ± 879.5 vs 963.3 ± 333.7, p-value = 0.008) CPNE1 gene showed the most significant corre-lation between CNAs and transcript levels (correcorre-lation
Figure 5 Genomic profile comparisons between colorectal cancer (CRC) subtypes (a) Gene-based copy number alteration (CNA) frequency difference between CRC subtypes Each dot represents the significance of CNA frequency difference between MSS and MSI-H subtypes of each gene (Fisher ’s exact test) Top dots indicate the -log 10 (p-value) of genes with CN gains, and bottom dots indicate the log 10 (p-value) of genes with CN losses (b) Comparison of gene expression differences between CRC subtypes Each dot represents the log2 scale of average expression fold-change (MSS/MSI-H) of each gene (two sample t-test, p-value < 0.05).
Table 2 Cancer genes showing differences in copy number aberration between CRC subtypes
Gene Symbol1 Frequency of CN Gain Frequency of CN Loss Gene expression profile
MSS 2 MSI-H 2 P-value MSS 2 MSI-H 2 P-value MSS 2 MSI-H 2 Fold-change (p-value) EGFR 0.62 0 0.0003 926+1088 369+197 2.51 (0.106) EXT1 0.69 0.06 0.00099 829+285 618+244 1.34 (0.053) GNAS 0.54 0 0.0011 16843+4876 13486+4555 1.25 (0.082) HOXA11 0.46 0 0.00361 ND3 ND3
-HOXA13 0.46 0 0.00361 368+364 496+318 0.74 (0.346) HOXA9 0.46 0 0.00361 1979+2078 2521+1705 0.79 (0.472) IKZF1 0.46 0 0.00361 ND3 ND3
-JAZF1 0.46 0 0.00361 153+128 145+43 1.05 (0.847) LHFP 0.54 0.06 0.0097 553+486 355+172 1.56 (0.202) MAFB 0.62 0 0.0003 648+687 711+351 0.91 (0.776) TOP1 0.54 0 0.0011 156+49 151+40 1.03 (0.768) MALT1 0.69 0 0.00007 258+79 334+105 0.77 (0.051)
1
The list of cancer gene from the Cancer Genome Project (http://www.sanger.ac.uk/genetics/CGP/).
2
The sample sizes of MSS and MSI-H are 13 and 16, respectively.
3
Trang 9coefficient, r2 = 0.7) CPNE1 gene regulates tumour
necrosis factor-alpha receptor signaling pathway and is
over-expressed in liver cancer [31,32], but is still poorly
investigated in CRC tumorigenesis
Discussion
This is a large-scale sporadic CRC study in an Asian
population, and our results showed that the
clinicopatho-logic features of MSI-H tumors were right-sided
predo-minant, poorly or mucinous diffenentiated, less advanced
disease and female predominant Similar to previous
stu-dies with Lynch syndrome [6,22], MSI-H in our case
ser-ies of sporadic CRC bear epigenetic change of MLH1
gene However, the clinical features are distinctly
differ-ent, and they tend to have older age onset of cancer and
female predominant For rectal cancer, the percentage of
MSI-H and MLH1 methylation was only 2.8% (9/401)
and 1% (4/401) respectively On the other hand,
right-sided colon cancer had, 16.3% and 11.2% MSI-H and
MLH1 methylation, respectively Therefore, dysfunction
of MMR proteins might play different roles in the
tumor-igenesis of colon cancer vs rectal cancer It is noteworthy
that all 22 samples with a BRAF (V599E) mutation were
MLH1 hypermethylated, whereas 29 of 51 tumors with
MLH1 hypermethylation did not have a BRAF mutation
These findings suggest that MLH1 hypermethylation
might be an early event, occurred prior to BRAF
muta-tion during CRC tumorigenesis
We have applied high-density SNP array to detect
copy number changes in the CRC cancer genome in the
Taiwanese population, and compared the CNA
frequen-cies between MSS and MSI-H subtypes Previous CRC
CN analyses primarily concerned with the Caucasian
genetic backgrounds and these studies were hampered
by the low-resolution of CGH array Although different
populations and technological resolutions were used in
this study, the overall CNV pattern was globally similar
to those from previous studies, indicating the
mechan-ism of CRC tumorigenesis of different ethnic
popula-tions might be similar Although EGFR CN gains were
commonly found in MSS tumors (64%), some MSI-H
tumors (14%) carried three or four gene copies Previous
studies have shown a small proportion of MSI-H tumors
harbor multiple CNAs and chromosome abnormalities
[17] Consistently, we also observed some MSI-H
tumors carried more than 1 Mb CNAs (Additional File
1), and 27.5% MSI-H tumors showed DNA aneuploidy
Studies showed that response predictors for CRC
patients using cetuximab, EGFR monoclonal antibody,
included K-ras/Braf mutation and EGFR gene CN, etc
[33,34] Further investigations are needed to clarify
whether MSI tumors might be resistant to cetuximab
for possible BRAF mutation or relatively low copy
num-ber of EGFR gene Among 12,012 tumor-expressed
transcripts, 514 genes showed significant CN gains or losses in MSS tumors, but 48% of them were not directly correlated with their expressional levels For example, 8/13 MSS and 0/16 MSI-H tumors have EGFR
CN gains; the expression fold-change of MSS/MSI-H group was 2.5 (962.4/368.8) but not significant (p-value
= 0.10), caused by large standard deviation of EGFR expression levels (Table 2) Besides CNVs, other geno-mic variants, including SNPs and Indels, and epigenogeno-mic modifications all can regulate transcript levels, so an integrated analysis are needed to interpret the transcript diversities between CRC subtypes
The identified CRC subtype-specific CN-altered genes should be seriously considered when investigating the mechanism of heterogeneous CRC tumorigenesis, and might be used as candidate markers in the drug therapy studies The major discrepancy, and argument, between our results and other studies was that the proportion of MSI-H in our study was only 6.4%, lower than that of previous reports [35-38] Selection bias and racial and/
or environmental factors might affect the MSI incidence
in CRCs Because rectal cancer is less likely to show MSI-H than colon cancer [39], a lower rate of MSI-H colorectal cancer will be reflected in population-based studies In studies without selection [39-41] incidence of MSI would be similar to our results
Additional material
Additional file 1: The size distribution of copy number variation in colorectal cancer CNVs were called by using Affymetrix Genotyping Console program based on the intensity data of Affymetrix SNP 6.0 array, and 20-probe criterion was used to filter out false-positive predictions The sizes of identified CN changes from MSS CRCs were majorly between 50 and 500 kb, and a quarter of these alterations were smaller than 100 kb.
Additional file 2: Genes showing copy number (CN) differences between MSS and MSI-H CRC cases 1,515 genes were found to have
CN frequency difference between MSS and MSI-H tumors using Fisher ’s exact tests (p-value < 0.05).
Additional file 3: The verification of EGFR copy number states of 48 CRC MSS and 48 MSI-H clinical samples qPCR approach was used to determine the EGFR CN states of 48 CRC MSS and 48 MSI-H samples Additional file 4: Differently-expressed transcripts between MSS and MSI-H CRC cases Among 24,526 transcripts of Illumina Human
Ref-8 gene expression array, 599 and 724 transcripts showed higher- or lower-expressions, respectively, in MSS tumors compared to MSI-H Additional file 5: Expression fold-changes between CRC subtypes in different populations There were subtle diversities in CRC transcript profiles between Caucasian and Han Chinese populations.
Additional file 6: The combined analysis of copy number alterations (CNAs) and gene expressions 1,515 genes showing different CNA frequencies between CRC subtypes, and 514 of them were expressed in these tumor tissues 271 of 514 genes (52%) show differential expressions between CRC MSS and MSI-H subtypes (two sample t-test with p-value < 0.05).
Additional file 7: The positive correlation between copy number and expression in CPNE1 gene The average CPNE1 expressional levels
of MSS group was higher then that of MSI-H group (p-value = 0.008),
Trang 10and the gene CNs were highly correlated to expressional levels (liner
regression correlation coefficient, r 2 = 0.7).
Acknowledgements
This project was supported by the Department of Health of Taiwan
(DOH99-TD-C-111-007; DOH99-TD-C-111-014), National Science Council grant of
Taiwan (NSC97-2314-B-010-019-MY2), Taipei-Veterans General Hospital
(V100E2-008) and the National Health Research Institutes, Taiwan.
Author details
1 Division of Molecular and Genomic Medicine, National Health Research
Institutes, Zhunan, Taiwan 2 Division of Colon and Rectal Surgery,
Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan.
3 Division of Hematology and Oncology, Taipei Veterans General Hospital,
Taipei, Taiwan.4Institute of Biomedical Sciences, Academia Sinica, Taipei,
Taiwan 5 Genome Research Center and Department of Life Sciences and
Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan.
Authors ’ contributions
CHL, JKL, SCC, SFT and WSC conceived of experiments; CHL, SCC, YHC and
HMC performed experiments; CHL, JKL, LHL and YTC provided and analyzed
data; all authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 19 November 2010 Accepted: 7 June 2011
Published: 7 June 2011
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